PMC 20140719 pmc.key 4850273 CC BY-NC-SA no 0 0 Xyloglucan Recognition by Gut Bacteria Tauzin et al. 10.1128/mBio.02134-15 4850273 27118585 mBio02134-15 e02134-15 2 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. surname:Tauzin;given-names:Alexandra S. surname:Kwiatkowski;given-names:Kurt J. surname:Orlovsky;given-names:Nicole I. surname:Smith;given-names:Christopher J. surname:Creagh;given-names:A. Louise surname:Haynes;given-names:Charles A. surname:Wawrzak;given-names:Zdzislaw surname:Brumer;given-names:Harry surname:Koropatkin;given-names:Nicole M. TITLE front 7 2016 0 Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:58:24Z Xyloglucan species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z Human ABSTRACT abstract_title_1 81 ABSTRACT ABSTRACT abstract 90 Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:22Z Polysaccharide utilization loci gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:41:37Z carbohydrates gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:30Z bacterial chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:56:33Z complex polysaccharide protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:20Z glycosidases experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:08Z biochemical, structural, and reverse genetic characterization protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:44:49Z cell surface glycan-binding proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:06Z xyloglucan utilization locus gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL species MESH: melaniev@ebi.ac.uk 2023-03-16T15:48:01Z Bacteroides ovatus taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:50:28Z vegetable chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:41Z polysaccharide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T21:46:06Z Biochemical analysis protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:44:54Z outer membrane-anchored proteins chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:34Z xyloglucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:43Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:41Z polysaccharide evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:50:03Z crystal structure protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:51:30Z bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:43Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:51:35Z tetradecasaccharide site SO: melaniev@ebi.ac.uk 2023-03-16T15:48:38Z carbohydrate-binding platform chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:41:40Z β-glucan structure_element SO: melaniev@ebi.ac.uk 2023-03-16T21:48:14Z tetra-modular evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:46:31Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:49:26Z tandem Ig-like folds chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:58:35Z XyG structure_element SO: melaniev@ebi.ac.uk 2023-03-16T15:49:16Z C-terminal domain evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:42:56Z affinities chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:43Z XyG experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:50:16Z reverse-genetic analysis protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:51:46Z oligosaccharides protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:59:07Z carbohydrate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:43Z XyG protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:58:56Z carbohydrate species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:51Z B. ovatus taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:50:28Z vegetable chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:52:04Z xyloglucans taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes ABSTRACT abstract_title_1 1947 IMPORTANCE ABSTRACT abstract 1958 The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:54:54Z bacteria species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:55:37Z complex polysaccharides taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:55:04Z plant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:14:06Z polysaccharides experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T15:55:51Z biochemical, crystallographic, and genetic insight protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:00Z surface glycan-binding proteins species MESH: melaniev@ebi.ac.uk 2023-03-16T15:48:01Z Bacteroides ovatus gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:55:59Z xyloglucan utilization locus gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:50:28Z vegetable chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:41Z polysaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:56:25Z complex polysaccharide taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:54:54Z bacteria INTRO title_1 2852 INTRODUCTION INTRO paragraph 2865 The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance. This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche. More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health. However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment. The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures. The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function. species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:03:40Z microbiota species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:03:30Z microbial taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:30Z bacterial taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:03:51Z Firmicutes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:04:02Z Actinobacteria chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:41:45Z carbohydrates species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:30Z bacterial taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:03:40Z microbiota species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:04:30Z complex carbohydrate taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:03:40Z microbiota species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:54:54Z bacteria taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:27Z glycan taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:03:51Z Firmicutes species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:41:11Z polysaccharide utilization loci gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T16:06:19Z starch utilization system complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T16:06:28Z Sus species MESH: melaniev@ebi.ac.uk 2023-03-16T16:06:08Z Bacteroides thetaiotaomicron complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T16:06:28Z Sus protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:05:57Z lipid-anchored protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:05:48Z endo-amylase protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:49Z SusG protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:06:34Z TonB-dependent transporter protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:06:38Z TBDT protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:58Z SusC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:07:03Z oligosaccharides protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:05:44Z starch-binding protein protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:07:24Z carbohydrate-binding lipoproteins protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:33Z SusE protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:44Z SusF protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:05:37Z exo-glucosidases protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:58Z SusA protein PR: melaniev@ebi.ac.uk 2023-03-16T16:08:08Z SusB chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:08:13Z glucose gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron species MESH: melaniev@ebi.ac.uk 2023-03-16T15:48:01Z Bacteroides ovatus gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:40Z reverse-genetic, biochemical, and structural studies gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL mbo0021627940001.jpg fig1 FIG fig_caption 5194 Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:11:10Z Xyloglucan species MESH: melaniev@ebi.ac.uk 2023-03-16T15:48:01Z Bacteroides ovatus gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:11:16Z xyloglucan utilization locus gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:11:20Z structures chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:52:04Z xyloglucans gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:12:52Z BoXyGUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:20Z glycosidases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:12:08Z GHs taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:12:17Z solanaceous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:12:23Z xyloglucan gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:12:37Z BtSus gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:12:52Z BoXyGUL gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:12:52Z BoXyGUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:52:04Z xyloglucans protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T16:13:18Z B protein PR: melaniev@ebi.ac.uk 2023-03-16T16:13:26Z GH5 INTRO paragraph 5909 We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). XyG variants (Fig. 1A) constitute up to 25% of the dry weight of common vegetables. Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface. gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human species MESH: melaniev@ebi.ac.uk 2023-03-16T16:25:03Z B. ovatus ATCC 8483 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:55:04Z plant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:41:52Z glycans chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:52:04Z xyloglucans chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:43Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:43Z XyG taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:25:25Z vegetables gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:25:36Z Sus locus gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:25:42Z xyloglucan utilization locus gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:26:24Z carbohydrate-binding, -hydrolyzing, and -importing proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:25:57Z glycoside hydrolases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:12:08Z GHs gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:25:37Z Sus locus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:26:10Z starch protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:12:08Z GHs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:59:58Z XyG INTRO paragraph 6811 In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. The genes coding for these proteins, sometimes referred to as “susE/F positioned,” display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins. As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T16:06:19Z starch utilization system species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron site SO: melaniev@ebi.ac.uk 2023-03-16T16:30:12Z starch-binding sites protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:30:21Z surface glycan-binding proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:30:34Z amylase protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:49Z SusG protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:33Z SusE protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:44Z SusF protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:31:00Z starch protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:33Z SusE protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:44Z SusF protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:49Z SusG site SO: melaniev@ebi.ac.uk 2023-03-16T16:31:05Z binding sites chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:14:34Z polysaccharide taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:58Z SusC protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:31:21Z susD gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:31:30Z susE/F protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:31:36Z putative protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:31:39Z lipoproteins gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:31:30Z susE/F gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:31:44Z carbohydrate-binding proteins complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T16:06:29Z Sus protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:27Z glycan gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:14:06Z polysaccharides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG INTRO paragraph 8045 We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B. ovatus. These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:42:46Z functional and structural characterization protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:42:51Z noncatalytic protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:42:55Z Bacova_02651 gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:43:00Z Bacova_02650 gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:43:05Z biochemical, structural, and reverse-genetic approaches chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:00:18Z XyG species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:27Z glycan taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:14:34Z polysaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:43:22Z xyloglucan RESULTS title_1 8734 RESULTS AND DISCUSSION RESULTS title_2 8757 SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:43:47Z cell-surface-localized, xyloglucan-specific binding proteins RESULTS paragraph 8841 SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:45:44Z Bacova_02651 species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:45:54Z SusD-like proteins gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:41:16Z Bacova_02650 gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:58Z SusC protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan RESULTS paragraph 9828 Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:47:57Z Immunofluorescence chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B ptm MESH: melaniev@ebi.ac.uk 2023-03-16T16:48:08Z lipidation protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:48:15Z cell-surface-localized endo-xyloglucanase species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus protein PR: melaniev@ebi.ac.uk 2023-03-16T16:13:26Z GH5 protein PR: melaniev@ebi.ac.uk 2023-03-16T16:48:42Z BoGH5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:48:31Z SusC-like TBDT gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL mbo0021627940002.jpg fig2 FIG fig_caption 10372 SGBP-A and SGBP-B visualized by immunofluorescence. Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. (D) FITC images of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:03Z immunofluorescence species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:07Z Overlay evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:51:11Z bright-field and FITC images species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T21:46:13Z Overlay evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:51:15Z bright-field and FITC images species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:51:19Z Bright-field image mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:51:35Z FITC images mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:51:31Z lacking protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG RESULTS paragraph 11064 In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:25:57Z glycoside hydrolases gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:12Z affinity PAGE experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:56:57Z isothermal titration calorimetry experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:05Z ITC protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:05Z xyloglucan-binding proteins evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:34:50Z affinity constant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:05Z Ka experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:12Z affinity PAGE protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:18Z hydroxyethyl cellulose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:24Z HEC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:29Z β(1 → 4)-glucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:32Z mixed-linkage β(1→3)/β(1→4)-glucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:39Z MLG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:44Z glucomannan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:51Z GM chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:55Z glucosyl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:58Z mannosyl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:14:34Z polysaccharide protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:48Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:24Z HEC protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:39Z MLG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:51Z GM protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:08Z SGBP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:58:03Z galactomannan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:58:09Z GGM chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:58:13Z starch chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:58:16Z carboxymethylcellulose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:58:20Z mucin protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:01Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:58:25Z XyG-specific GHs gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:30Z PUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:39Z MLG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:57:51Z GM chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:58:09Z GGM site SO: melaniev@ebi.ac.uk 2023-03-16T16:58:32Z carbohydrate-binding modules protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:12:09Z GHs gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T16:58:35Z xyloglucan protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T16:58:39Z -B mbo0021627940003.jpg fig3 FIG fig_caption 12557 SGBP-A and SGBP-B preferentially bind xyloglucan. Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. All samples were loaded on the same gel next to the BSA controls; thin black lines indicate where intervening lanes were removed from the final image for both space and clarity. The percentage of polysaccharide incorporated into each native gel is displayed. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:00:22Z xyloglucan experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T17:00:26Z Affinity electrophoresis protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B protein PR: melaniev@ebi.ac.uk 2023-03-16T17:00:34Z BSA protein PR: melaniev@ebi.ac.uk 2023-03-16T17:00:34Z BSA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide RESULTS paragraph 12960 The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. Fig. 1A). The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:13Z endo-xyloglucanase gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein PR: melaniev@ebi.ac.uk 2023-03-16T16:48:42Z BoGH5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:41:57Z glucosyl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:42:01Z xylogluco-oligosaccharides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:42:05Z XyGOs structure_element SO: melaniev@ebi.ac.uk 2023-03-16T21:48:31Z Glc4 backbone structure_element SO: melaniev@ebi.ac.uk 2023-03-16T21:48:36Z variable side-chain galactosylation chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T21:46:18Z controlled digestion and fractionation experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T21:46:22Z size exclusion chromatography chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:42:08Z oligosaccharides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:05Z ITC protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T21:49:23Z Glc8 backbone evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:42:55Z affinities chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T21:49:29Z Glc4 backbone protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:08:44Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:42:57Z affinities evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:54Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:36Z affinity structure_element SO: melaniev@ebi.ac.uk 2023-03-16T18:36:41Z minimal repeating unit evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:14:34Z polysaccharide protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:50:04Z dimer structure_element SO: melaniev@ebi.ac.uk 2023-03-16T21:49:33Z minimal repeat chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:12Z affinity PAGE chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:42:13Z oligosaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:42:18Z cellotetraose protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:42:25Z cellohexaose evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:09:08Z affinity chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:12Z hexasaccharide gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T21:46:26Z X-ray crystallography protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T21:46:39Z SGPB-B complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T21:49:37Z oligosaccharide-complex forms tab1.xml tab1 TABLE table_caption 14796 Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:34:04Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T18:33:57Z isothermal titration calorimetry tab1.xml tab1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><colgroup span="1"><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/></colgroup><thead><tr><th rowspan="2" colspan="1">Carbohydrate</th><th colspan="2" rowspan="1"><italic>K_a</italic> (M⁻¹)<hr/></th><th colspan="2" rowspan="1">Δ<italic>G</italic> (kcal ⋅ mol⁻¹)<hr/></th><th colspan="2" rowspan="1">Δ<italic>H</italic> (kcal ⋅ mol⁻¹)<hr/></th><th colspan="2" rowspan="1"><italic>T</italic>Δ<italic>S</italic> (kcal ⋅ mol⁻¹)<hr/></th></tr><tr><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">XyG<xref ref-type="table-fn" rid="ngtab1.2">^b</xref></td><td rowspan="1" colspan="1">(4.4 ± 0.1) × 10⁵</td><td rowspan="1" colspan="1">(5.7 ± 0.2) × 10⁴</td><td rowspan="1" colspan="1">−7.7</td><td rowspan="1" colspan="1">−6.5</td><td rowspan="1" colspan="1">−14 ± 3</td><td rowspan="1" colspan="1">−14 ± 2</td><td rowspan="1" colspan="1">−6.5</td><td rowspan="1" colspan="1">−7.6</td></tr><tr><td rowspan="1" colspan="1">XyGO₂<xref ref-type="table-fn" rid="ngtab1.3">^c</xref></td><td rowspan="1" colspan="1">3.0 × 10⁵</td><td rowspan="1" colspan="1">2.0 × 10⁴</td><td rowspan="1" colspan="1">−7.5</td><td rowspan="1" colspan="1">−5.9</td><td rowspan="1" colspan="1">−17.2</td><td rowspan="1" colspan="1">−17.6</td><td rowspan="1" colspan="1">−9.7</td><td rowspan="1" colspan="1">−11.7</td></tr><tr><td rowspan="1" colspan="1">XyGO₁</td><td rowspan="1" colspan="1">NB<xref ref-type="table-fn" rid="ngtab1.4">^d</xref></td><td rowspan="1" colspan="1">(2.4 ± 0.1) × 10³</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−4.6</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−4.4 ± 0.2</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">0.2</td></tr><tr><td rowspan="1" colspan="1">Cellohexaose</td><td rowspan="1" colspan="1">568.0 ± 291.0</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−3.8</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−16 ± 8</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−12.7</td><td rowspan="1" colspan="1">NB</td></tr><tr><td rowspan="1" colspan="1">Cellotetraose</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td></tr></tbody></table> 14919 Carbohydrate Ka (M−1) ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) SGBP-A SGBP-B SGBP-A SGBP-B SGBP-A SGBP-B SGBP-A SGBP-B XyGb (4.4 ± 0.1) × 105 (5.7 ± 0.2) × 104 −7.7 −6.5 −14 ± 3 −14 ± 2 −6.5 −7.6 XyGO2c 3.0 × 105 2.0 × 104 −7.5 −5.9 −17.2 −17.6 −9.7 −11.7 XyGO1 NBd (2.4 ± 0.1) × 103 NB −4.6 NB −4.4 ± 0.2 NB 0.2 Cellohexaose 568.0 ± 291.0 NB −3.8 NB −16 ± 8 NB −12.7 NB Cellotetraose NB NB NB NB NB NB NB NB tab1.xml tab1 TABLE table_footnote 15427 Shown are average values ± standard errors from two independent titrations, unless otherwise indicated. tab1.xml tab1 TABLE table_footnote 15532 Binding thermodynamics for XyG based on the concentration of the binding unit, XyGO2. tab1.xml tab1 TABLE table_footnote 15618 Values from a single titration. tab1.xml tab1 TABLE table_footnote 15650 NB, no binding observed. RESULTS title_2 15675 SGBP-A is a SusD homolog with an extensive glycan-binding platform. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD site SO: melaniev@ebi.ac.uk 2023-03-16T18:37:53Z glycan-binding platform RESULTS paragraph 15743 As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:42:28Z structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:42:35Z apo protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:15Z Rwork evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:40Z Rfree residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:51Z 28 to 546 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T18:43:55Z “SusD-like” protein fold structure_element SO: melaniev@ebi.ac.uk 2023-03-16T18:44:02Z tetratrico-peptide repeat structure_element SO: melaniev@ebi.ac.uk 2023-03-16T18:44:05Z TPR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:44:09Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T18:44:12Z overlays species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD protein PR: melaniev@ebi.ac.uk 2023-03-16T18:44:26Z BtSusD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:44:47Z root mean square deviation evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:45:02Z RMSD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T18:45:13Z Cocrystallization protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T18:45:27Z substrate complex evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:45:31Z structure evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:17Z Rwork evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:43Z Rfree residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:46:04Z 36 to 546 site SO: melaniev@ebi.ac.uk 2023-03-16T21:48:27Z binding-site protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:18Z XyG binding protein complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T18:42:50Z SGBP-A:XyGO2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T18:46:14Z superimposes protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:42:35Z apo evidence DUMMY: melaniev@ebi.ac.uk 2023-06-15T08:35:30Z structure evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:45:04Z RMSD site SO: melaniev@ebi.ac.uk 2023-03-16T18:46:24Z ligand-binding site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:46:27Z conserved protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A site SO: melaniev@ebi.ac.uk 2023-03-16T18:46:33Z aromatic platform chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG site SO: melaniev@ebi.ac.uk 2023-03-16T18:47:02Z site protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:46:53Z amylose mbo0021627940004.jpg fig4 FIG fig_caption 17245 Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. The apo structure is color ramped from blue to red. An omit map (2σ) for XyGO2 (orange and red sticks) is displayed. (B) Close-up view of the omit map as in panel A, rotated 90° clockwise. (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. (D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites. The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. Potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:56:14Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A gene GENE: melaniev@ebi.ac.uk 2023-03-16T18:56:21Z Bacova_02651 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T18:56:18Z Overlay protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:42:35Z apo chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:56:26Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:42:35Z apo evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:47:03Z structure evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:24:59Z omit map chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:24:59Z omit map experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T18:56:34Z Overlay protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:21Z XyGO2 protein PR: melaniev@ebi.ac.uk 2023-03-16T18:44:26Z BtSusD chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:56:41Z maltoheptaose site SO: melaniev@ebi.ac.uk 2023-03-16T18:56:50Z glycan-binding site protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD site SO: melaniev@ebi.ac.uk 2023-03-16T18:56:58Z glycan-binding sites site SO: melaniev@ebi.ac.uk 2023-03-16T18:56:50Z glycan-binding site evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:57:12Z protein structures site SO: melaniev@ebi.ac.uk 2023-03-16T18:57:08Z xyloglucan-binding site protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:57:18Z glucose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:57:16Z xylose residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:57:21Z X1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:57:24Z X2 tab2.xml tab2 TABLE table_caption 18298 X-ray data collection and refinement statistics tab2.xml tab2 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><colgroup span="1"><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/></colgroup><thead><tr><th rowspan="2" colspan="1">Parameter</th><th colspan="4" rowspan="1">Value(s) for<xref ref-type="table-fn" rid="ngtab2.1">^a</xref>:<hr/></th></tr><tr><th rowspan="1" colspan="1">SGBP-A <italic>apo</italic></th><th rowspan="1" colspan="1">SGBP-A/XyGO₂</th><th rowspan="1" colspan="1">SGBP-B/XyGO₂</th><th rowspan="1" colspan="1">SGBP-B (CD)/XyGO₂</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">PDB ID no.</td><td rowspan="1" colspan="1">5E75</td><td rowspan="1" colspan="1">5E76</td><td rowspan="1" colspan="1">5E7G</td><td rowspan="1" colspan="1">5E7H</td></tr><tr><td rowspan="1" colspan="1">Resolution (Å)</td><td rowspan="1" colspan="1">21.48–1.36 (1.409–1.36)</td><td rowspan="1" colspan="1">56.13–2.3 (2.382–2.3)</td><td rowspan="1" colspan="1">39.19–2.37 (2.455–2.37)</td><td rowspan="1" colspan="1">30.69–1.57 (1.626–1.570)</td></tr><tr><td rowspan="1" colspan="1">Space group</td><td rowspan="1" colspan="1">P2₁</td><td rowspan="1" colspan="1">I422</td><td rowspan="1" colspan="1">R32</td><td rowspan="1" colspan="1">P6₁22</td></tr><tr><td rowspan="1" colspan="1">Unit cell dimensions, <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td rowspan="1" colspan="1">52.8, 81.4, 57.7; β = 107.85°</td><td rowspan="1" colspan="1">131.5, 131.5, 188</td><td rowspan="1" colspan="1">207.4, 207.4, 117.9</td><td rowspan="1" colspan="1">87.1, 87.1, 201.6</td></tr><tr><td rowspan="1" colspan="1">No. of reflections</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    Total</td><td rowspan="1" colspan="1">355,272 (26,772)</td><td rowspan="1" colspan="1">1,068,014 (102,923)</td><td rowspan="1" colspan="1">324,544 (32,355)</td><td rowspan="1" colspan="1">1,366,812 (129,645)</td></tr><tr><td rowspan="1" colspan="1">    Unique</td><td rowspan="1" colspan="1">99,136 (9,762)</td><td rowspan="1" colspan="1">36,775 (3,625)</td><td rowspan="1" colspan="1">39,362 (3,898)</td><td rowspan="1" colspan="1">62,808 (6,068)</td></tr><tr><td rowspan="1" colspan="1">Multiplicity</td><td rowspan="1" colspan="1">3.6 (2.7)</td><td rowspan="1" colspan="1">29.0 (28.4)</td><td rowspan="1" colspan="1">8.2 (8.3)</td><td rowspan="1" colspan="1">21.8 (21.4)</td></tr><tr><td rowspan="1" colspan="1">Completeness (%)</td><td rowspan="1" colspan="1">99.71 (98.82)</td><td rowspan="1" colspan="1">99.63 (99.42)</td><td rowspan="1" colspan="1">99.96 (100.00)</td><td rowspan="1" colspan="1">98.4 (96.98)</td></tr><tr><td rowspan="1" colspan="1">Mean <italic>I</italic>/σ〈<italic>I</italic>〉</td><td rowspan="1" colspan="1">15.57 (2.29)</td><td rowspan="1" colspan="1">24.93 (6.71)</td><td rowspan="1" colspan="1">20.98 (2.36)</td><td rowspan="1" colspan="1">38.52 (5.03)</td></tr><tr><td rowspan="1" colspan="1">Wilson B-factor</td><td rowspan="1" colspan="1">11.91</td><td rowspan="1" colspan="1">31.14</td><td rowspan="1" colspan="1">43.91</td><td rowspan="1" colspan="1">17.86</td></tr><tr><td rowspan="1" colspan="1"><italic>R</italic>_merge</td><td rowspan="1" colspan="1">0.04759 (0.4513)</td><td rowspan="1" colspan="1">0.1428 (0.7178)</td><td rowspan="1" colspan="1">0.09159 (1.197)</td><td rowspan="1" colspan="1">0.05559 (0.7748)</td></tr><tr><td rowspan="1" colspan="1">CC_1/2<xref ref-type="table-fn" rid="ngtab2.2">^b</xref></td><td rowspan="1" colspan="1">0.999 (0.759)</td><td rowspan="1" colspan="1">0.999 (0.982)</td><td rowspan="1" colspan="1">0.999 (0.794)</td><td rowspan="1" colspan="1">1.000 (0.933)</td></tr><tr><td rowspan="1" colspan="1">CC*<xref ref-type="table-fn" rid="ngtab2.3">^c</xref></td><td rowspan="1" colspan="1">1.000 (0.929)</td><td rowspan="1" colspan="1">1.000 (0.995)</td><td rowspan="1" colspan="1">1.000 (0.941)</td><td rowspan="1" colspan="1">1.000 (0.982)</td></tr><tr><td rowspan="1" colspan="1"><italic>R</italic>_work</td><td rowspan="1" colspan="1">0.1468 (0.2597)</td><td rowspan="1" colspan="1">0.2178 (0.2788)</td><td rowspan="1" colspan="1">0.1975 (0.3018)</td><td rowspan="1" colspan="1">0.1560 (0.2008)</td></tr><tr><td rowspan="1" colspan="1"><italic>R</italic>_free</td><td rowspan="1" colspan="1">0.1738 (0.2632)</td><td rowspan="1" colspan="1">0.2482 (0.2978)</td><td rowspan="1" colspan="1">0.2260 (0.3219)</td><td rowspan="1" colspan="1">0.1712 (0.2019)</td></tr><tr><td rowspan="1" colspan="1">No. of non-hydrogen atoms</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    All</td><td rowspan="1" colspan="1">4,562</td><td rowspan="1" colspan="1">4,319</td><td rowspan="1" colspan="1">3,678</td><td rowspan="1" colspan="1">2,328</td></tr><tr><td rowspan="1" colspan="1">    Macromolecules</td><td rowspan="1" colspan="1">4,079</td><td rowspan="1" colspan="1">3,974</td><td rowspan="1" colspan="1">3,425</td><td rowspan="1" colspan="1">1,985</td></tr><tr><td rowspan="1" colspan="1">    Ligands</td><td rowspan="1" colspan="1">39</td><td rowspan="1" colspan="1">116</td><td rowspan="1" colspan="1">127</td><td rowspan="1" colspan="1">25</td></tr><tr><td rowspan="1" colspan="1">    Water</td><td rowspan="1" colspan="1">444</td><td rowspan="1" colspan="1">229</td><td rowspan="1" colspan="1">126</td><td rowspan="1" colspan="1">318</td></tr><tr><td rowspan="1" colspan="1">No. of protein residues</td><td rowspan="1" colspan="1">506</td><td rowspan="1" colspan="1">492</td><td rowspan="1" colspan="1">446</td><td rowspan="1" colspan="1">260</td></tr><tr><td rowspan="1" colspan="1">RMSD</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    Bond length (Å)</td><td rowspan="1" colspan="1">0.008</td><td rowspan="1" colspan="1">0.007</td><td rowspan="1" colspan="1">0.005</td><td rowspan="1" colspan="1">0.009</td></tr><tr><td rowspan="1" colspan="1">    Bond angle (°)</td><td rowspan="1" colspan="1">1.15</td><td rowspan="1" colspan="1">0.96</td><td rowspan="1" colspan="1">0.87</td><td rowspan="1" colspan="1">1.18</td></tr><tr><td rowspan="1" colspan="1">Ramachandran statistics</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    Favored (%)</td><td rowspan="1" colspan="1">98</td><td rowspan="1" colspan="1">95</td><td rowspan="1" colspan="1">97</td><td rowspan="1" colspan="1">98</td></tr><tr><td rowspan="1" colspan="1">    Outliers (%)</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0.41</td><td rowspan="1" colspan="1">0.23</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">    Clash score</td><td rowspan="1" colspan="1">0.5</td><td rowspan="1" colspan="1">2.13</td><td rowspan="1" colspan="1">0.86</td><td rowspan="1" colspan="1">1.27</td></tr><tr><td rowspan="1" colspan="1">Avg B-factors</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    All</td><td rowspan="1" colspan="1">16.1</td><td rowspan="1" colspan="1">53.2</td><td rowspan="1" colspan="1">53</td><td rowspan="1" colspan="1">25.4</td></tr><tr><td rowspan="1" colspan="1">    Macromolecules</td><td rowspan="1" colspan="1">15.2</td><td rowspan="1" colspan="1">53.5</td><td rowspan="1" colspan="1">52.5</td><td rowspan="1" colspan="1">22.9</td></tr><tr><td rowspan="1" colspan="1">    Ligands</td><td rowspan="1" colspan="1">24.7</td><td rowspan="1" colspan="1">61</td><td rowspan="1" colspan="1">71.1</td><td rowspan="1" colspan="1">47</td></tr><tr><td rowspan="1" colspan="1">    Solvent</td><td rowspan="1" colspan="1">24.4</td><td rowspan="1" colspan="1">42.9</td><td rowspan="1" colspan="1">47.6</td><td rowspan="1" colspan="1">39</td></tr></tbody></table> 18346 Parameter Value(s) fora: SGBP-A apo SGBP-A/XyGO2 SGBP-B/XyGO2 SGBP-B (CD)/XyGO2 PDB ID no. 5E75 5E76 5E7G 5E7H Resolution (Å) 21.48–1.36 (1.409–1.36) 56.13–2.3 (2.382–2.3) 39.19–2.37 (2.455–2.37) 30.69–1.57 (1.626–1.570) Space group P21 I422 R32 P6122 Unit cell dimensions, a, b, c (Å) 52.8, 81.4, 57.7; β = 107.85° 131.5, 131.5, 188 207.4, 207.4, 117.9 87.1, 87.1, 201.6 No. of reflections     Total 355,272 (26,772) 1,068,014 (102,923) 324,544 (32,355) 1,366,812 (129,645)     Unique 99,136 (9,762) 36,775 (3,625) 39,362 (3,898) 62,808 (6,068) Multiplicity 3.6 (2.7) 29.0 (28.4) 8.2 (8.3) 21.8 (21.4) Completeness (%) 99.71 (98.82) 99.63 (99.42) 99.96 (100.00) 98.4 (96.98) Mean I/σ〈I〉 15.57 (2.29) 24.93 (6.71) 20.98 (2.36) 38.52 (5.03) Wilson B-factor 11.91 31.14 43.91 17.86 Rmerge 0.04759 (0.4513) 0.1428 (0.7178) 0.09159 (1.197) 0.05559 (0.7748) CC1/2b 0.999 (0.759) 0.999 (0.982) 0.999 (0.794) 1.000 (0.933) CC*c 1.000 (0.929) 1.000 (0.995) 1.000 (0.941) 1.000 (0.982) Rwork 0.1468 (0.2597) 0.2178 (0.2788) 0.1975 (0.3018) 0.1560 (0.2008) Rfree 0.1738 (0.2632) 0.2482 (0.2978) 0.2260 (0.3219) 0.1712 (0.2019) No. of non-hydrogen atoms     All 4,562 4,319 3,678 2,328     Macromolecules 4,079 3,974 3,425 1,985     Ligands 39 116 127 25     Water 444 229 126 318 No. of protein residues 506 492 446 260 RMSD     Bond length (Å) 0.008 0.007 0.005 0.009     Bond angle (°) 1.15 0.96 0.87 1.18 Ramachandran statistics     Favored (%) 98 95 97 98     Outliers (%) 0 0.41 0.23 0     Clash score 0.5 2.13 0.86 1.27 Avg B-factors     All 16.1 53.2 53 25.4     Macromolecules 15.2 53.5 52.5 22.9     Ligands 24.7 61 71.1 47     Solvent 24.4 42.9 47.6 39 tab2.xml tab2 TABLE table_footnote 20239 Numbers in parentheses are for the highest-resolution shell. tab2.xml tab2 TABLE table_footnote 20300 CC1/2, Pearson correlation coefficient between the average intensities of each subset. tab2.xml tab2 TABLE table_footnote 20387 CC*, Pearson correlation coefficient for correlation between the observed data set and true signal. RESULTS paragraph 20487 Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. Fig. 1). Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site. Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:17Z glucosyl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:02:43Z ligand electron density chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:25Z α(1→6)-linked xylosyl evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:02:47Z electron density chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:02:17Z oligosaccharide site SO: melaniev@ebi.ac.uk 2023-03-16T19:02:32Z binding site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:03:44Z W82 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:03:51Z W283 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:03:58Z W306 site SO: melaniev@ebi.ac.uk 2023-03-16T19:03:27Z flat platform chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:03:37Z β-glucan site SO: melaniev@ebi.ac.uk 2023-03-16T19:03:30Z platform mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:12Z W82A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:19Z W283A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:27Z W306A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:03:19Z mutant protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:49:42Z SGBP-A* protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:45:29Z completely devoid of XyG affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:12Z W82A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:04:32Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:27Z W306A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:04Z substitution protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:45:00Z abolishes XyG binding chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:30Z ligand residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:04:50Z R65 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:04:56Z N83 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:05:03Z S308 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:05:10Z Glc5 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:05:16Z Glc3 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:04:40Z saccharide-binding data chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:05:13Z XyG residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:05:24Z Y84 site SO: melaniev@ebi.ac.uk 2023-06-15T08:28:18Z hydrophobic interface chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:49:40Z xylosyl residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:05:32Z Xyl1 tab3.xml tab3 TABLE table_caption 21867 Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:05Z ITC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG tab3.xml tab3 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><colgroup span="1"><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/></colgroup><thead><tr><th rowspan="2" colspan="1">Protein name</th><th colspan="2" rowspan="1"><italic>K_a</italic><hr/></th><th rowspan="2" colspan="1">Δ<italic>G</italic> (kcal ⋅ mol⁻¹)</th><th rowspan="2" colspan="1">Δ<italic>H</italic> (kcal ⋅ mol⁻¹)</th><th rowspan="2" colspan="1"><italic>T</italic>Δ<italic>S</italic> (kcal ⋅ mol⁻¹)</th></tr><tr><th rowspan="1" colspan="1">Fold change<xref ref-type="table-fn" rid="ngtab3.2">^b</xref></th><th rowspan="1" colspan="1">M⁻¹</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">SGBP-A(W82A W283A W306A)</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td></tr><tr><td rowspan="1" colspan="1">SGBP-A(W82A)<xref ref-type="table-fn" rid="ngtab3.3">^c</xref></td><td rowspan="1" colspan="1">4.9</td><td rowspan="1" colspan="1">9.1 × 10⁴</td><td rowspan="1" colspan="1">−6.8</td><td rowspan="1" colspan="1">−6.3</td><td rowspan="1" colspan="1">0.5</td></tr><tr><td rowspan="1" colspan="1">SGBP-A(W306)</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(230–489)</td><td rowspan="1" colspan="1">0.7</td><td rowspan="1" colspan="1">(8.6 ± 0.20) × 10⁴</td><td rowspan="1" colspan="1">−6.7</td><td rowspan="1" colspan="1">−14.9 ± 0.1</td><td rowspan="1" colspan="1">−8.2</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(Y363A)</td><td rowspan="1" colspan="1">19.7</td><td rowspan="1" colspan="1">(2.9 ± 0.10) × 10³</td><td rowspan="1" colspan="1">−4.7</td><td rowspan="1" colspan="1">−18.1 ± 0.1</td><td rowspan="1" colspan="1">−13.3</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(W364A)</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">Weak</td><td rowspan="1" colspan="1">Weak</td><td rowspan="1" colspan="1">Weak</td><td rowspan="1" colspan="1">Weak</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(F414A)</td><td rowspan="1" colspan="1">3.2</td><td rowspan="1" colspan="1">(1.80 ± 0.03) × 10⁴</td><td rowspan="1" colspan="1">−5.8</td><td rowspan="1" colspan="1">−11.4 ± 0.1</td><td rowspan="1" colspan="1">−5.6</td></tr></tbody></table> 21985 Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:05:33Z ΔH evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:05:42Z TΔS protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:12Z W82A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:19Z W283A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:27Z W306A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:12Z W82A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:02Z SGBP-A residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:03:58Z W306 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:07:33Z 230–489 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:47:43Z Y363A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:47:55Z W364A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:49:54Z F414A tab3.xml tab3 TABLE table_footnote 22478 Shown are average values ± standard deviations from two independent titrations, unless otherwise indicated. Binding thermodynamics are based on the concentration of the binding unit, XyGO2. Weak binding represents a Ka of <500 M−1. ND, not determined; NB, no binding. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka tab3.xml tab3 TABLE table_footnote 22749 Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:34:04Z wild-type evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:06:02Z xyloglucan tab3.xml tab3 TABLE table_footnote 22835 Values from a single titration. RESULTS title_2 22867 SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:08:13Z glycan-binding domain RESULTS paragraph 22952 The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). Such domains are not typically involved in carbohydrate binding. Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6 Å for 253 aligned residues. While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:50:03Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:15:21Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:15:29Z in complex with chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:17Z Rwork evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:43Z Rfree residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:15:34Z 34 to 489 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:47:27Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:15:50Z tandem immunoglobulin (Ig)-like domains structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:02Z A structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:05Z B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:08Z C structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:21Z xyloglucan-binding domain structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:17:18Z D structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:27Z A structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:31Z B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:34Z C structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:43Z β-sandwich folds structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:46Z B residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:16:49Z 134 to 230 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:16:53Z C residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:16:55Z 231 to 313 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:17:22Z superimposed structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:17:37Z A residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:17:40Z 34 to 133 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:18:01Z RMSDs structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:18:07Z These domains structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:18:15Z β-sandwich domains protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:18:18Z GH13 enzymes protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:18:21Z cyclodextrin glucanotransferase species MESH: melaniev@ebi.ac.uk 2023-03-16T19:18:24Z Geobacillus stearothermophilus structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:18:28Z Such domains chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:06:17Z carbohydrate experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:18:31Z visual inspection protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:18:34Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:18:39Z A structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:18:42Z B experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:12Z affinity PAGE chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:06:29Z XyG experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:18:49Z production mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:18:56Z fused domains C and D protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:19:04Z 230 to 489 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:19:23Z xyloglucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:15:21Z full-length structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:19:50Z D protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:19:54Z XyG-binding proteins protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:19:58Z xylan-binding protein protein PR: melaniev@ebi.ac.uk 2023-03-16T19:20:01Z Bacova_04391 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:07:21Z xylan gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:20:08Z structure-based alignment evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:45:04Z RMSD protein PR: melaniev@ebi.ac.uk 2023-03-16T19:20:21Z Bacova_04391 site SO: melaniev@ebi.ac.uk 2023-03-16T19:20:25Z binding site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:20:14Z W241 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:20:17Z Y404 site SO: melaniev@ebi.ac.uk 2023-03-16T19:20:28Z XyGO binding site protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:20:34Z opposing, clamp-like arrangement structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:20:36Z these residues protein PR: melaniev@ebi.ac.uk 2023-03-16T19:20:39Z Bacova_04391 site SO: melaniev@ebi.ac.uk 2023-03-16T19:20:42Z planar surface arrangement structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:20:45Z residues chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B mbo0021627940005.jpg fig5 FIG fig_caption 25179 Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. Prolines between domains are indicated as spheres. An omit map (2σ) for XyGO2 is displayed to highlight the location of the glycan-binding site. (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B gene GENE: melaniev@ebi.ac.uk 2023-03-16T19:24:43Z Bacova_02650 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:15:21Z Full-length evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:24:48Z structure protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B residue_name SO: melaniev@ebi.ac.uk 2023-03-16T19:24:52Z Prolines evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:24:59Z omit map chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 site SO: melaniev@ebi.ac.uk 2023-03-16T18:56:50Z glycan-binding site protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:25:09Z A structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:25:13Z B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:25:16Z C structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:25:25Z Ig-like domain species MESH: melaniev@ebi.ac.uk 2023-03-16T19:25:29Z G. stearothermophilus protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:25:34Z cyclodextrin glucanotransferase residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:25:38Z 375 to 493 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:25:44Z overlay protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B protein PR: melaniev@ebi.ac.uk 2023-03-16T19:25:48Z Bacova_04391 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:24:59Z omit map chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 site SO: melaniev@ebi.ac.uk 2023-03-16T18:57:08Z xyloglucan-binding site protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:25:52Z glucose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:25:55Z xylose residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:25:59Z X1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:26:02Z X2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:26:07Z X3 RESULTS paragraph 26120 Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396 Å2. Domains A, B, and C do not pack against each other. Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig. 5A). Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:31:26Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:31:30Z C structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:31:34Z D structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:31:37Z A structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:31:41Z B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:31:44Z C structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:31:48Z five-residue linkers residue_name SO: melaniev@ebi.ac.uk 2023-03-16T19:31:52Z proline structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:31:55Z middle residue residue_name SO: melaniev@ebi.ac.uk 2023-03-16T19:31:58Z proline structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:32:02Z Ig-like domains protein PR: melaniev@ebi.ac.uk 2023-03-16T19:32:05Z Bacova_04391 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:32:09Z starch-binding proteins protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:33Z SusE protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:44Z SusF site SO: melaniev@ebi.ac.uk 2023-03-16T19:32:14Z glycan binding site protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:32:22Z eight-residue linker protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:32:49Z lipidated residue_name SO: melaniev@ebi.ac.uk 2023-03-16T19:32:55Z Cys residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:32:58Z C28 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:33:01Z first β-strand structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:33:21Z A protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:33:26Z outer membrane proteins complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T19:33:29Z Sus-like systems structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:33:33Z 10- to 20-amino-acid flexible linker protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:32:49Z lipidated residue_name SO: melaniev@ebi.ac.uk 2023-03-16T19:33:38Z Cys protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:33:41Z outer membrane-anchored protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T19:33:44Z endo-xyloglucanase protein PR: melaniev@ebi.ac.uk 2023-03-16T16:48:42Z BoGH5 gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:33:51Z 100-amino-acid, all-β-strand structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:33:54Z N-terminal module structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:33:57Z flexible linker structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:34:00Z catalytic module RESULTS paragraph 27551 XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. Fig. 1A). The backbone is flat, with less of the “twisted-ribbon” geometry observed in some cello- and xylogluco-oligosaccharides. The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:46:03Z binds to structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:46:07Z D protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B site SO: melaniev@ebi.ac.uk 2023-03-16T19:46:10Z concave interface structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:46:14Z β-sheet structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:46:17Z loops structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:46:21Z β-strands chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:46:25Z glucosyl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:49:40Z xylosyl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:46:42Z density chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:46:47Z cello- and xylogluco-oligosaccharides site SO: melaniev@ebi.ac.uk 2023-03-16T18:46:34Z aromatic platform residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:46:59Z W330 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:47:07Z W364 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:47:14Z Y363 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:47:21Z glucosyl protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:47:24Z longer site SO: melaniev@ebi.ac.uk 2023-03-16T19:47:27Z platform protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:47:36Z glucosyl mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:47:43Z Y363A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:47:46Z site-directed mutant protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:49Z SGBP-B evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:44Z XyG mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:47:55Z W364A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:47:59Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:48:08Z lacks XyG binding chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:48:15Z β-glucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:49:40Z xylosyl experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T16:57:05Z ITC protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:08:04Z cellohexaose residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:49:03Z F414 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:49:40Z xylosyl residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:05:16Z Glc3 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:49:21Z Q407 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:49:40Z xylosyl residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:05:33Z Xyl1 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:49:54Z F414A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:49:59Z mutant protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:35:07Z Ka chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:08:17Z glycan structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:50:04Z residues site SO: melaniev@ebi.ac.uk 2023-03-16T19:50:07Z binding site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:50:14Z Y369 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:50:22Z E412 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG protein PR: melaniev@ebi.ac.uk 2023-06-15T08:38:53Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-06-15T08:39:01Z xyloglucan experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:50:33Z solved evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:50:03Z crystal structure mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:50:38Z fused CD domains protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:15:29Z in complex with chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:17Z Rwork evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:43:43Z Rfree residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:50:46Z 230 to 489 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T19:50:49Z CD domains protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:50:52Z truncated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:15:21Z full-length experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:50:57Z superimpose evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:45:04Z RMSD site SO: melaniev@ebi.ac.uk 2023-03-16T19:51:01Z glycan-binding residues evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:51:05Z density chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:51:08Z density evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:08:41Z X-ray data evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:51:14Z crystal structures chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:51:11Z sugar RESULTS title_2 30050 SGBP-A and SGBP-B have distinct, coordinated functions in vivo. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B RESULTS paragraph 30114 The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5 mg/ml of the reported carbohydrate. Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). It is unknown whether this is because cultures were not normalized by the starting optical density (OD) or viable cells or reflects a minor defect for glucose utilization. The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose. The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. Therefore, we limit our discussion to those mutants that displayed the most obvious defects in growth on particular substrates. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:08:58Z XyG species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:09:09Z XyG experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:08Z in-frame deletion and complementation mutant species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:11Z growth experiments chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:57:19Z glucose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:57:16Z carbohydrate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:57:24Z glucose evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:50Z lag time evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:25:13Z lag times chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:57:21Z carbohydrate evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:52Z lag time chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:57:27Z glucose gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:30Z deleted evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:58:46Z lag chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:57:32Z glucose protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:34:04Z wild-type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:59:09Z lags chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:09:23Z glucose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:57:58Z glucose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:58:01Z xylose mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:53Z ΔXyGUL protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:25:15Z lag times chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:58:03Z xylose evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:52Z lag time chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:58:05Z xylose protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T19:58:28Z Complementation mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:34:04Z wild-type chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T19:58:24Z xyloglucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:58:14Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 mbo0021627940006.jpg fig6 FIG fig_caption 32273 Growth of select XyGUL mutants on xyloglucan and oligosaccharides. B. ovatus mutants were created in a thymidine kinase deletion (Δtdk) mutant as described previously. SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed. Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT Δtdk strain. Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material. gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:01:10Z xyloglucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:01:14Z oligosaccharides species MESH: melaniev@ebi.ac.uk 2023-03-16T21:40:58Z B. ovatus mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:01:17Z thymidine kinase deletion mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:01:24Z SGBP-A* gene GENE: melaniev@ebi.ac.uk 2023-03-16T20:01:28Z Bacova_02651 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:12Z W82A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:19Z W283A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:27Z W306A protein PR: melaniev@ebi.ac.uk 2023-03-16T20:01:33Z GH9 gene GENE: melaniev@ebi.ac.uk 2023-03-16T20:01:36Z Bacova_02649 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:01:44Z glucose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:01:50Z xylose evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:52Z lag time chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:01:53Z glucose protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:01:56Z carbohydrate protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:49:49Z ΔSBGP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T18:34:04Z wild-type evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:25:15Z lag times RESULTS paragraph 33503 The ΔSGBP-A (ΔBacova_02651) strain (cf. Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. Similarly, the function of SGBP-A extends beyond glycan binding. Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). The specific glycan signal that upregulates BoXyGUL is currently unknown. From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL. However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells. mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:07:26Z ΔBacova_02651 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:09:56Z XyG complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T16:06:29Z Sus species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:07:49Z ΔsusD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:07:42Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:07:54Z starch chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:07:56Z malto-oligosaccharides gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T20:07:59Z complementation mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:07:50Z ΔsusD mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:08:03Z SusD* protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:08:06Z triple site-directed mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:49:53Z W96A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:49:56Z W320A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:49:58Z Y296A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:08:15Z ablates glycan binding species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:08:21Z malto-oligosaccharides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:08:25Z starch gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:41:21Z sus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:08:27Z maltose protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T20:08:31Z Complementation mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:08:34Z SGBP-A* mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:12Z W82A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:20Z W283A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:04:27Z W306A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:09:06Z not bind chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:36Z XyGOs mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:08:37Z SGBP-A* protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:10:09Z carbohydrate protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:09:22Z maltose mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:07:50Z ΔsusD mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:09:24Z SusD* chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:09:26Z starch evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:26:19Z lag phase mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:09:29Z SGBP-A* evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:52Z lag time chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T20:09:32Z xyloglucan protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:12:53Z BoXyGUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:09:39Z SGBP-A* chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG RESULTS paragraph 35481 Intriguingly, the ΔSGBP-B strain (ΔBacova_02650) (cf. Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. However, growth of the ΔSGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca. 10-fold more weakly than XyGO2 and XyG (Fig. 6; Table 1). As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:23:05Z ΔBacova_02650 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T19:57:38Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T19:57:45Z Δtdk mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:40Z oligo- and polysaccharides protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:44Z oligosaccharides protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:23:11Z double mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:23:14Z crippled protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:03Z SGBP-A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T20:23:17Z deletion of protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:23:20Z SGBP-A* mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:52Z lag time chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:45:22Z XyGO2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:04Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:50Z XyGOs protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B protein PR: melaniev@ebi.ac.uk 2023-03-16T20:23:32Z BtSusE protein PR: melaniev@ebi.ac.uk 2023-03-16T20:23:34Z BtSusF gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:25:37Z Sus locus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:53Z starch chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:43:56Z malto-oligosaccharides protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:04Z SGBP-A protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:23:37Z catalytically feeble protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T20:23:41Z endo-xyloglucanase protein PR: melaniev@ebi.ac.uk 2023-03-16T20:23:44Z GH9 protein PR: melaniev@ebi.ac.uk 2023-03-16T16:13:27Z GH5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T20:24:01Z combined deletion of the genes encoding protein PR: melaniev@ebi.ac.uk 2023-03-16T21:47:32Z GH9 gene GENE: melaniev@ebi.ac.uk 2023-03-16T20:23:56Z Bacova_02649 protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T18:36:33Z XyGO1 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:24:05Z ΔGH9 RESULTS paragraph 37123 The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. Our previous work demonstrates that B. ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript. Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin. We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT. In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:18:27Z SGBP-A* mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:18:30Z SGBP-A* mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:26Z ΔSGBP-B protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:18:33Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:17Z lag chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:49:45Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:18:37Z xylogluco-oligosaccharides evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:17Z lag protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:18:54Z malto-oligosaccharide species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T20:24:17Z lag chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:44:01Z xyloglucan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:02Z SGBPs species MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:52Z B. ovatus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:19:03Z glucose gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:19:05Z glucose gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL protein PR: melaniev@ebi.ac.uk 2023-03-16T16:13:27Z GH5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs protein PR: melaniev@ebi.ac.uk 2023-03-16T16:13:27Z GH5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:19:12Z xyloglucan protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:10:53Z SGBP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:19:16Z oligosaccharides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:15Z XyGUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:04Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:19:25Z TBDT gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:19:38Z BtSus protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:18Z SusD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:19:27Z TBDT protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:59Z SusC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:25Z glycan mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:07:50Z ΔsusD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:48:22Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:19:45Z starch mutant MESH: melaniev@ebi.ac.uk 2023-03-16T20:07:50Z ΔsusD mutant MESH: melaniev@ebi.ac.uk 2023-03-16T21:19:54Z SusD* protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:20:06Z transcriptional activator gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:19:42Z sus operon protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:04Z SGBP-A protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:23Z TBDT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T16:51:43Z ΔSGBP-A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:20:20Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:20:23Z xyloglucan complex_assembly GO: melaniev@ebi.ac.uk 2023-03-16T16:06:29Z Sus experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T21:20:16Z elimination of protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:33Z SusE protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:45Z SusF chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:20:26Z starch protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:20:28Z xylogluco-oligosaccharides RESULTS title_2 39216 Conclusions. RESULTS paragraph 39229 The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans. The human gut, in particular, is a densely packed ecosystem with hundreds of species, in which there is potential for both competition and synergy in the utilization of different substrates. Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. Thus, understanding glycan capture at the cell surface is fundamental to explaining, and eventually predicting, how the carbohydrate content of the diet shapes the gut community structure as well as its causative health effects. Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:41:31Z microorganisms chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:23:32Z glycans species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:23:38Z glycans protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:25:57Z glycoside hydrolases chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:24:00Z oligosaccharides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:23:42Z surface glycan binding proteins gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:12:53Z BoXyGUL taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:50:28Z vegetable chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:23:54Z xyloglucan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:25:57Z glycoside hydrolases protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:23:47Z TonB-dependent transporters RESULTS paragraph 40417 A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported. On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity. For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics. Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent. Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:28Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:29:12Z TBDTs protein PR: melaniev@ebi.ac.uk 2023-03-16T16:06:59Z SusC gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:29:12Z TBDTs taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:30Z iron-targeting TBDTs taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:29:42Z Proteobacteria protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:33Z glycan-importing TBDTs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:37Z SGBP protein PR: melaniev@ebi.ac.uk 2023-03-16T21:29:33Z BtSusC protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:40Z TBDT protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:19Z SusD protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:43Z SGBP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:29Z glycan gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL species MESH: melaniev@ebi.ac.uk 2023-03-16T21:41:02Z Capnocytophaga canimorsus protein PR: melaniev@ebi.ac.uk 2023-03-16T16:07:19Z SusD protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:04Z SGBP-A chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:12:13Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:12:05Z XyG chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:29Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:47Z SusC-like TBDTs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:51Z SusD-like SGBPs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:29Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:53Z TBDT protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:29:51Z carbohydrate protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:29:12Z TBDTs taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:56Z SGBP gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:29:59Z nanO protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:45:59Z SusC-like TBDT gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL species MESH: melaniev@ebi.ac.uk 2023-03-16T21:30:06Z B. fragilis chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:29:53Z monosaccharide species MESH: melaniev@ebi.ac.uk 2023-03-16T21:41:05Z E. coli gene GENE: melaniev@ebi.ac.uk 2023-03-16T16:31:21Z susD gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:41:26Z nanU gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:30:01Z nanU gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:30:03Z BT1762 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:30:12Z fructan-targeting SusD-like protein species MESH: melaniev@ebi.ac.uk 2023-03-16T16:08:28Z B. thetaiotaomicron chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:29:55Z fructans protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:30:14Z SusD-like SGBP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:29Z glycan taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:29:12Z TBDTs gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs gene GENE: melaniev@ebi.ac.uk 2023-03-16T21:30:23Z carbohydrate utilization containing TBDT [CUT] loci taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:31Z bacterial taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes RESULTS paragraph 42733 Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length. The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL. This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency. Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown. However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:32:20Z SusD-like proteins protein PR: melaniev@ebi.ac.uk 2023-03-16T15:49:04Z SGBP-A protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:32:23Z glycan-binding lipoproteins gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:16Z XyGUL taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T21:32:51Z Bacteroides protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T16:12:09Z GHs gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:31Z PUL protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs protein PR: melaniev@ebi.ac.uk 2023-03-16T21:32:57Z BtSusE protein PR: melaniev@ebi.ac.uk 2023-03-16T21:33:00Z BtSusF gene GENE: melaniev@ebi.ac.uk 2023-03-16T15:48:16Z XyGUL protein PR: melaniev@ebi.ac.uk 2023-03-16T15:48:50Z SGBP-B chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T15:50:42Z polysaccharide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:29Z glycan taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:54:55Z bacteria protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:29Z glycan protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T15:47:03Z SGBPs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:33:08Z carbohydrates structure_element SO: melaniev@ebi.ac.uk 2023-03-16T21:33:10Z carbohydrate-binding motifs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:33:12Z carbohydrate RESULTS paragraph 44381 In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes. The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms. A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T21:34:13Z surface-glycan binding proteins chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T21:34:16Z carbohydrates taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:47:40Z Bacteroidetes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:54:55Z bacteria chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T17:14:06Z polysaccharides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:29Z glycan chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:30Z glycan species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T15:54:55Z bacteria species MESH: melaniev@ebi.ac.uk 2023-03-16T15:41:08Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T16:03:40Z microbiota METHODS title_1 45063 MATERIALS AND METHODS METHODS title_2 45085 Protein production and purification. METHODS paragraph 45122 The gene fragments corresponding to Bacova_02650 (encoding SGBP-B residues 34 to 489) and Bacova_02651 (encoding SGBP-A residues 28 to 546) were amplified from Bacteroidetes ovatus ATCC 8483 genomic DNA by PCR using forward primers, including NdeI restriction sites, and reverse primers, including XhoI. The gene products were ligated into a modified version of pET-28a (EMD Biosciences) containing a recombinant tobacco etch virus (rTEV) protease recognition site (pET-28aTEV) preceding an N-terminal 6-His tag for affinity purification. The expression vector (pET-28TEV) containing SGBP-B was used for subsequent cloning of the domains A (residues 34 to 133), B (residues 134 to 229), and CD (residues 230 to 489). The pET-28TEV vector expressing residues 28 to 546 of SGBP-A was utilized for carbohydrate-binding experiments and crystallization of the apo structure. To obtain crystals of SGBP-A with XyGO2, the DNA sequence coding for residues 36 to 546 was PCR amplified from genomic DNA for ligation-independent cloning into the pETite N-His vector (Lucigen, Madison, WI) according to the manufacturer’s instructions. The N-terminal primer for pETite N-His insertion contained a TEV cleavage site immediately downstream of the complementary 18-bp overlap (encoding the His tag) to create a TEV-cleavable His-tagged protein. The site-directed mutants of SGBP-A and SGBP-B in pET-28TEV were created using the QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The sequences of all primers to generate these constructs are displayed in Table S1 in the supplemental material. METHODS paragraph 46758 The plasmids containing the SGBP-A and SGBP-B genes were transformed into Escherichia coli BL21(DE3) or Rosetta(DE3) cells. For native protein expression, cells were cultured in Terrific Broth containing kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml) at 37°C to the mid-exponential phase (A600 of ≈0.6), induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and then incubated for 2 days at 16°C or 1 day at 20°C. Cells were harvested by centrifugation and frozen at −80°C prior to protein purification. For selenomethionine-substituted SGBP-B, the pET-28TEV-SGBP-B plasmid was transformed into E. coli Rosetta(DE3)/pLysS and plated onto LB supplemented with kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml). After 16 h of growth at 37°C, colonies were harvested from the plates, used to inoculate 100 ml of M9 minimal medium supplemented with kanamycin (30 µg/ml) and chloramphenicol (20 µg/ml), and then grown at 37°C for 16 h. This overnight culture was used to inoculate a 2-liter baffled flask containing 1 liter of Molecular Dimensions SelenoMet premade medium supplemented with 50 ml of the recommended sterile nutrient mix, chloramphenicol, and kanamycin. Cultures were grown at 37°C to an A600 of ≈0.45 before adjusting the temperature to 20°C and supplementing each flask with 100 mg each of l-lysine, l-threonine, and l-phenylalanine and 50 mg each of l-leucine, l-isoleucine, l-valine, and l-selenomethionine. After 20 additional minutes of growth, the cells were induced with 0.5 mM IPTG, and cultures were grown for an additional 48 h. METHODS paragraph 48382 For the purification of native and selenomethionine-substituted protein, cells were thawed and lysed via sonication in His buffer (25 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.5) and purified via immobilized nickel affinity chromatography (His-Trap; GE Healthcare) using a gradient of 20 to 300 mM imidazole, according to the manufacturer’s instructions. The His tag was removed by incubation with TEV protease (1:100 molar ratio relative to protein) at room temperature for 2 h and then overnight at 4°C while being dialyzed against His buffer. The cleaved protein was then repurified via nickel affinity chromatography to remove undigested target protein, the cleaved His tag, and His-tagged TEV protease. Purified proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0) prior to crystallization and concentrated using Vivaspin 15 (10,000-molecular-weight-cutoff [MWCO]) centrifugal concentrators (Vivaproducts, Inc.). METHODS title_2 49330 Glycans. METHODS paragraph 49339 Xyloglucan from tamarind seed, β-glucan from barley, and konjac glucomannan were purchased from Megazyme. Starch, guar, and mucin were purchased from Sigma. Hydroxyethyl cellulose was purchased from AMRESCO. Carboxymethyl cellulose was purchased from Acros Organics. Xylogluco-oligosaccharides XyGO1 and XyGO2 for biophysical analyses were prepared from tamarind seed XyG according to the method of Martinez-Fleites et al. with minor modifications. XyGO2 for cocrystallization was purchased from Megazyme (O-XGHDP). METHODS title_2 49858 Affinity gel electrophoresis. METHODS paragraph 49888 Affinity PAGE was performed as described previously, with minor modification. Various polysaccharides were used at a concentration of 0.05 to 0.1% (wt/vol), and electrophoresis was carried out for 90 min at room temperature in native 10% (wt/vol) polyacrylamide gels. BSA was used as noninteracting negative-control protein. METHODS title_2 50214 ITC. METHODS paragraph 50219 Isothermal titration calorimetry (ITC) of glycan binding by the SGPB-A mutants was performed using the TA Nano isothermal titration calorimeter calibrated to 25°C. Proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0), and sugars were prepared using the dialysis buffer. The protein (45 to 50 µM) was placed in the sample cell, and the syringe was loaded with 2.5 to 4 mg/ml XyG polysaccharide. Following an initial injection of 0.5 µl, 26 subsequent injections of 2 µl were performed with stirring at 350 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Nano Analyze software. All other ITC experiments were performed using a MicroCal VP-ITC titration calorimeter calibrated to 25°C. Proteins were dialyzed into 20 mM HEPES–100 mM NaCl (pH 7.0), and polysaccharides were prepared using the dialysis buffer. Proteins (micromolar concentrations) were placed in the sample cell, and a first injection of 2 µl was performed followed by 24 subsequent injections of 10 µl of 2 to 20 mM oligosaccharide (cellotetraose, cellohexaose, XyGO1, or XyGO2) or 1 to 2.5 mg/ml XyG polysaccharide. The solution was stirred at 280 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Origin software program. METHODS title_2 51512 DSC. METHODS paragraph 51517 Structural integrity of the SGBP-B mutants was verified by differential scanning calorimetry (DSC). DSC studies were performed on a MicroCal VP-DSC (Malvern Instruments). Experiments were carried out in 50 mM HEPES (pH 7.0) at a scan rate of 60°C/h. All samples (40 µM protein) were degassed for 7 min with gentle stirring under vacuum prior to being loaded into the calorimeter. Background excess thermal power scans were obtained with buffer in both the sample and reference cells and subtracted from the scans for each sample solution to generate excess heat capacity versus temperature thermograms. METHODS paragraph 52126 The melting temperature decreased from 57.8 ± 0.9°C for the wild-type SGBP-B protein to 54.6 ± 0.1°C for the Y363A mutant, 54.2 ± 0.1°C for the W364A mutant, and 52 ± 1°C for the F414A mutant. All proteins were therefore in their stable folded state for the ITC measurements (see Fig. S9 in the supplemental material). METHODS title_2 52454 Bacteroides ovatus mutagenesis. METHODS paragraph 52486 Gene deletions and complementations were performed via allelic exchange in a Bacteroides ovatus thymidine kinase gene (Bacova_03071) deletion (Δtdk) derivative strain of ATCC 8483 using the vector pExchange-tdk, as previously described. Primers for the construction of B. ovatus mutants are listed in Table S1. The B. ovatus Δtdk strain and the B. ovatus ΔXyGUL mutant were a generous gift from Eric Martens, University of Michigan Medical School. METHODS title_2 52946 Bacteroides growth experiments. METHODS paragraph 52978 All Bacteroides ovatus culturing was performed in a Coy anaerobic chamber (85% N2, 10% H2, 5% CO2) at 37°C. Prior to growth on minimal medium plus the carbohydrates indicated (Fig. 6; see Fig. S8 in the supplemental material), each strain was grown for 16 h from a freezer stock in tryptone-yeast extract-glucose (TYG) medium and then back diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml glucose, as previously described. After growth for 24 h, cultures were back-diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml of glucose, xylose, XyG, XyGO1, or XyGO2. Growth experiments were performed in replicates of 12 (glucose, xylose, and xyloglucan) or 5 (XyGO1 and XyGO2) as 200-µl cultures in 96-well plates. Plates were loaded into a Biostack automated plate handling device coupled to a Powerwave HT absorbance reader (both devices from Biotek Instruments, Winooski, VT). Absorbance at 600 nm (A600; i.e., optical density at 600 nm [OD600]) was measured for each well at 20-min intervals. Data were processed using Gen5 software (BioTek) and Microsoft Excel. Growth was quantified in each assay by first identifying a minimum time point (Amin) at which A600 had increased by 15% over a baseline reading taken during the first 500 min of incubation. Next, we identified the time point at which A600 reached its maximum (Amax) immediately after exponential growth. The growth rate for each well was defined by (Amax − Amin)/(Tmax − Tmin), where Tmax and Tmin are the corresponding time values for each absorbance. To account for variations in inoculum density, for each strain, the lag time (Tmin) on glucose was subtracted from the lag time for the substrate of interest; in all cases, cultures had shorter lag times on glucose than other glycans. METHODS title_2 54783 Immunofluorescence. METHODS paragraph 54803 Custom rabbit antibodies to recombinant SGBP-A and SGBP-B were generated by Cocalico Biologicals, Inc. (Reamstown, PA). The B. ovatus ATCC 8483 Δtdk and ΔSGBP-B strains were grown in 1 ml minimal Bacteroides medium supplemented with 5 mg/ml tamarind xyloglucan to an A600 of ≈0.6 and then harvested via centrifugation (7,000 × g for 3 min) and washed twice with phosphate-buffered saline (PBS). Cells were resuspended in 0.25 ml PBS, and 0.75 ml of 6% formalin in PBS was added. Cells were incubated with rocking at 20°C for 1.5 h and then washed twice with PBS. Cells were resuspended in 0.5 to 1 ml blocking solution (2% goat serum, 0.02% NaN3 in PBS) and incubated for 16 h at 4°C. Cells were centrifuged and resuspended in 0.5 ml of a 1/100 dilution of custom rabbit antibody sera in blocking solution and incubated by rocking for 2 h at 20°C. Cells were washed with PBS and then resuspended in 0.4 ml of a 1/500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) in blocking solution and incubated with rocking for 1 h at 20°C. Cells were washed three times with an excess of PBS and then resuspended in 20 µl of PBS plus 1 µl of ProLong Gold antifade (Life Technologies). Cells were spotted on 0.8% agarose pads and imaged at the Center for Live Cell Imaging at the University of Michigan Medical School, using an Olympus IX70 inverted confocal microscope. Images were processed with Metamorph Software. METHODS title_2 56256 Crystallization and data collection. METHODS paragraph 56293 All X-ray diffraction data for both native and selenomethionine-substituted protein crystals were collected at the Life Science Consortium (LS-CAT) at the Advance Photon Source at Argonne National Laboratory in Argonne, IL. The native protein SGBP-B (residues 34 to 489) was concentrated to an A280 of 12.25 prior to crystallization and mixed with 10 mM XyGO2 (Megazyme, O-XGHDP). Hanging drop vapor diffusion was performed against mother liquor consisting of 1.1 to 1.5 M ammonium sulfate and 30 to 70 mM sodium cacodylate (pH 6.5). To decrease crystal nucleation, 0.3 ml of paraffin oil was overlaid on top of 0.5 ml of mother liquor yielding diffraction-quality crystals within 2 weeks. Selenomethionine-substituted crystals of SGBP-B were generated using the same conditions as the native crystals. Crystals of the truncated SGBP-B (domains CD, residues 230 to 489) were obtained by mixing concentrated protein (A600 of 20.6) with 10 mM XyGO2 for hanging drop vapor diffusion against a solution containing 2 M sodium formate and 0.1 M sodium acetate (pH 4.6). All SGBP-B crystals were flash-frozen prior to data collection by briefly soaking in a solution of 80% mother liquor–20% glycerol plus 10 mM xylogluco-oligosaccharide. Data were processed and scaled using HKL2000 and Scalepack. SAD phasing from a selenomethionine-substituted protein crystals was used to determine the structure of SGBP-B. The AutoSol and Autobuild algorithms within the Phenix suite of programs were used to locate and refine the selenium positions and automatically build an initial model of the protein structure, respectively. Successive rounds of manual model building and refinement in Coot and Phenix, respectively, were utilized to build a 2.7-Å model of the selenomethionine-substituted protein, which then was placed in the unit cell of the native data set. Additional rounds of manual model building and refinement were performed to complete the 2.37-Å structure of SGBP-B with XyGO2. The structure of the truncated protein (CD domains, residues 230 to 489) was solved via molecular replacement with Phaser using the CD domains of the full-length protein as a model. METHODS paragraph 58465 The native protein SGBP-A (residues 28 to 546) was concentrated to an A280 of 28.6 and crystallized via hanging drop vapor diffusion from the Morpheus crystal screen (Molecular Dimensions). Crystals formed in well A1 (30 mM MgCl2, 30 mM CaCl2, 20% polyethylene glycol [PEG 500], 10% PEG 20K, 0.1 M imidazole-MES [morpholinethanesulfonic acid], pH 6.5), and were flash-frozen in liquid nitrogen without additional cryoprotectant. The truncated SGBP-A (residues 36 to 546) concentrated to an A280 of 38.2 yielded crystals with 10 mM XyGO2 via hanging drop vapor diffusion against 1.2 to 1.8 M sodium citrate (pH 6.15 to 6.25), and were flash-frozen in a cryoprotectant solution of 80% mother liquor–20% ethylene glycol with the glycan. Data were processed and scaled using HKL2000 and Scalepack. The structure of the apo protein was solved via molecular replacement with BALBES using the homologous structure PDB 3JYS, followed by successive rounds of automatic and manual model building with Autobuild and Coot. The structure of SGBP-A with XyGO2 was solved via molecular replacement with Phaser and refined with Phenix. X-data collection and refinement statistics are presented in Table 2. SUPPL title_1 59664 SUPPLEMENTAL MATERIAL SUPPL footnote 59686 Citation Tauzin AS, Kwiatkowski KJ, Orlovsky NI, Smith CJ, Creagh AL, Haynes CA, Wawrzak Z, Brumer H, Koropatkin NM. 2016. Molecular dissection of xyloglucan recognition in a prominent human gut symbiont. mBio 7(2):e02134-15. doi:10.1128/mBio.02134-15. REF title 59939 REFERENCES 107 118 surname:Mazmanian;given-names:SK surname:Liu;given-names:CH surname:Tzianabos;given-names:AO surname:Kasper;given-names:DL 10.1016/j.cell.2005.05.007 16009137 REF Cell ref 122 2005 59950 An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system 62 surname:Sommer;given-names:F surname:Nookaew;given-names:I surname:Sommer;given-names:N surname:Fogelstrand;given-names:P surname:Bäckhed;given-names:F 10.1186/s13059-015-0614-4 25887251 REF Genome Biol ref 16 2015 60046 Site-specific programming of the host epithelial transcriptome by the gut microbiota 1241214 surname:Ridaura;given-names:VK surname:Faith;given-names:JJ surname:Muehlbauer;given-names:MJ surname:Ilkayeva;given-names:O surname:Semenkovich;given-names:CF surname:Funai;given-names:K surname:Hayashi;given-names:DK surname:Lyle;given-names:BJ surname:Martini;given-names:MC surname:Ursell;given-names:LK surname:Clemente;given-names:JC surname:Van Treuren;given-names:W surname:Rey;given-names:FE surname:Cheng;given-names:J surname:Duncan;given-names:AE surname:Kau;given-names:AL surname:Griffin;given-names:NW surname:Lombard;given-names:V surname:Henrissat;given-names:B surname:Bain;given-names:JR 10.1126/science.1241214 REF Science ref 341 2013 60131 Gut microbiota from twins discordant for obesity modulate metabolism in mice 979 984 surname:Bäckhed;given-names:F surname:Manchester;given-names:JK surname:Semenkovich;given-names:CF surname:Gordon;given-names:JI 10.1073/pnas.0605374104 17210919 REF Proc Natl Acad Sci U S A ref 104 2007 60208 Mechanisms underlying the resistance to diet-induced obesity in germ-free mice 1635 1638 surname:Eckburg;given-names:PB surname:Bik;given-names:EM surname:Bernstein;given-names:CN surname:Purdom;given-names:E surname:Dethlefsen;given-names:L surname:Sargent;given-names:M surname:Gill;given-names:SR surname:Nelson;given-names:KE surname:Relman;given-names:DA 10.1126/science.1110591 15831718 REF Science ref 308 2005 60287 Diversity of the human intestinal microbial flora 357 360 surname:Ding;given-names:T surname:Schloss;given-names:PD 10.1038/nature13178 24739969 REF Nature ref 509 2014 60337 Dynamics and associations of microbial community types across the human body 497 504 surname:El Kaoutari;given-names:A surname:Armougom;given-names:F surname:Gordon;given-names:JI surname:Raoult;given-names:D surname:Henrissat;given-names:B 10.1038/nrmicro3050 23748339 REF Nat Rev Microbiol ref 11 2013 60414 The abundance and variety of carbohydrate-active enzymes in the human gut microbiota e156 surname:Xu;given-names:J surname:Mahowald;given-names:MA surname:Cordum;given-names:H surname:Van Brunt;given-names:A surname:Kim;given-names:K surname:Fulton;given-names:RS surname:Fulton;given-names:LA surname:Clifton;given-names:SW surname:Wilson;given-names:RK surname:Knight;given-names:RD surname:Gordon;given-names:JI surname:Ley;given-names:RE surname:Lozupone;given-names:CA surname:Hamady;given-names:M surname:Martens;given-names:EC surname:Henrissat;given-names:B surname:Coutinho;given-names:PM surname:Minx;given-names:P surname:Latreille;given-names:P 10.1371/journal.pbio.0050156 17579514 REF PLoS Biol ref 5 2007 60499 Evolution of symbiotic bacteria in the distal human intestine 2074 2076 surname:Xu;given-names:J surname:Bjursell;given-names:MK surname:Himrod;given-names:J surname:Deng;given-names:S surname:Carmichael;given-names:LK surname:Chiang;given-names:HC surname:Hooper;given-names:LV surname:Gordon;given-names:JI 10.1126/science.1080029 12663928 REF Science ref 299 2003 60561 A genomic view of the human-Bacteroides thetaiotaomicron symbiosis 3851 3865 surname:Martens;given-names:EC surname:Kelly;given-names:AG surname:Tauzin;given-names:AS surname:Brumer;given-names:H 10.1016/j.jmb.2014.06.022 25026064 REF J Mol Biol ref 426 2014 60628 The devil lies in the details: how variations in polysaccharide fine-structure impact the physiology and evolution of gut microbes e1001221 surname:Martens;given-names:EC surname:Lowe;given-names:EC surname:Gordon;given-names:JI surname:Chiang;given-names:H surname:Pudlo;given-names:NA surname:Wu;given-names:M surname:McNulty;given-names:NP surname:Abbott;given-names:DW surname:Henrissat;given-names:B surname:Gilbert;given-names:HJ surname:Bolam;given-names:DN 10.1371/journal.pbio.1001221 22205877 REF PLoS Biol ref 9 2011 60759 Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts 5609 5616 surname:Tancula;given-names:E surname:Feldhaus;given-names:MJ surname:Bedzyk;given-names:LA surname:Salyers;given-names:AA 1512196 REF J Bacteriol ref 174 1992 60849 Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron 5365 5372 surname:Shipman;given-names:JA surname:Berleman;given-names:JE surname:Salyers;given-names:AA 10.1128/JB.182.19.5365-5372.2000 10986238 REF J Bacteriol ref 182 2000 60965 Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron 7206 7211 surname:Shipman;given-names:JA surname:Cho;given-names:KH surname:Siegel;given-names:HA surname:Salyers;given-names:AA 10572122 REF J Bacteriol ref 181 1999 61093 Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron 200 215 surname:Koropatkin;given-names:NM surname:Smith;given-names:TJ 10.1016/j.str.2009.12.010 20159465 REF Structure ref 18 2010 61224 SusG: A unique cell-membrane-associated alpha-amylase from a prominent human gut symbiont targets complex starch molecules 1105 1115 surname:Koropatkin;given-names:NM surname:Martens;given-names:EC surname:Gordon;given-names:JI surname:Smith;given-names:TJ 10.1016/j.str.2008.03.017 18611383 REF Structure ref 16 2008 61347 Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices 823 830 surname:Reeves;given-names:AR surname:D’Elia;given-names:JN surname:Frias;given-names:J surname:Salyers;given-names:AA 8550519 REF J Bacteriol ref 178 1996 61449 A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch 34614 34625 surname:Cameron;given-names:EA surname:Maynard;given-names:MA surname:Smith;given-names:CJ surname:Smith;given-names:TJ surname:Koropatkin;given-names:NM surname:Martens;given-names:EC 10.1074/jbc.M112.397380 22910908 REF J Biol Chem ref 287 2012 61573 Multidomain carbohydrate-binding proteins involved in Bacteroides thetaiotaomicron starch metabolism 7173 7179 surname:D’Elia;given-names:JN surname:Salyers;given-names:AA 8955399 REF J Bacteriol ref 178 1996 61674 Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch 19786 19791 surname:Hehemann;given-names:JH surname:Kelly;given-names:AG surname:Pudlo;given-names:NA surname:Martens;given-names:EC surname:Boraston;given-names:AB 10.1073/pnas.1211002109 23150581 REF Proc Natl Acad Sci U S A ref 109 2012 61800 Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes 165 169 surname:Cuskin;given-names:F surname:Lowe;given-names:EC surname:Rogowski;given-names:A surname:Hamilton;given-names:BS surname:Chen;given-names:R surname:Tolbert;given-names:TJ surname:Piens;given-names:K surname:Bracke;given-names:D surname:Vervecken;given-names:W surname:Hakki;given-names:Z surname:Speciale;given-names:G surname:Munōz-Munōz;given-names:JL surname:Temple;given-names:MJ surname:Day;given-names:A surname:Pena;given-names:MJ surname:McLean;given-names:R surname:Suits;given-names:MD surname:Boraston;given-names:AB surname:Atherly;given-names:T surname:Ziemer;given-names:CJ surname:Williams;given-names:SJ surname:Davies;given-names:GJ surname:Abbott;given-names:DW surname:Zhu;given-names:Y surname:Martens;given-names:EC surname:Gilbert;given-names:HJ surname:Cameron;given-names:EA surname:Pudlo;given-names:NA surname:Porter;given-names:NT surname:Urs;given-names:K surname:Thompson;given-names:AJ surname:Cartmell;given-names:A 10.1038/nature13995 25567280 REF Nature ref 517 2015 61932 Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism 7481 surname:Rogowski;given-names:A surname:Briggs;given-names:JA surname:Rogers;given-names:TE surname:Thompson;given-names:P surname:Hawkins;given-names:AR surname:Yadav;given-names:MP surname:Henrissat;given-names:B surname:Martens;given-names:EC surname:Dupree;given-names:P surname:Gilbert;given-names:HJ surname:Bolam;given-names:DN surname:Mortimer;given-names:JC surname:Tryfona;given-names:T surname:Terrapon;given-names:N surname:Lowe;given-names:EC surname:Basle;given-names:A surname:Morland;given-names:C surname:Day;given-names:AM surname:Zheng;given-names:H 10.1038/ncomms8481 26112186 REF Nat Commun ref 6 2015 62009 Glycan complexity dictates microbial resource allocation in the large intestine 498 502 surname:Larsbrink;given-names:J surname:Rogers;given-names:TE surname:Creagh;given-names:AL surname:Haynes;given-names:CA surname:Kelly;given-names:AG surname:Cederholm;given-names:SN surname:Davies;given-names:GJ surname:Martens;given-names:EC surname:Brumer;given-names:H surname:Hemsworth;given-names:GR surname:McKee;given-names:LS surname:Tauzin;given-names:AS surname:Spadiut;given-names:O surname:Klinter;given-names:S surname:Pudlo;given-names:NA surname:Urs;given-names:K surname:Koropatkin;given-names:NM 10.1038/nature12907 24463512 REF Nature ref 506 2014 62089 A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes 133 150 surname:McDougall;given-names:GJ surname:Morrison;given-names:IM surname:Stewart;given-names:D surname:Hillman;given-names:JR REF J Sci Food Agric ref 70 1996 62178 Plant cell walls as dietary fibre: range, structure, processing and function 526 542 surname:Schultink;given-names:A surname:Liu;given-names:L surname:Zhu;given-names:L surname:Pauly;given-names:M 10.3390/plants3040526 REF Plants ref 3 2014 62255 Structural diversity and function of xyloglucan sidechain substituents e01441-01414 surname:Cameron;given-names:EA surname:Kwiatkowski;given-names:KJ surname:Lee;given-names:BH surname:Hamaker;given-names:BR surname:Koropatkin;given-names:NM surname:Martens;given-names:EC 10.1128/mBio.01441-14 25205092 REF mBio ref 5 2014 62326 Multifunctional nutrient-binding proteins adapt human symbiotic bacteria for glycan competition in the gut by separately promoting enhanced sensing and catalysis 647 655 surname:Terrapon;given-names:N surname:Lombard;given-names:V surname:Gilbert;given-names:HJ surname:Henrissat;given-names:B 10.1093/bioinformatics/btu716 25355788 REF Bioinformatics ref 31 2015 62488 Automatic prediction of polysaccharide utilization loci in Bacteroidetes species 24673 24677 surname:Martens;given-names:EC surname:Koropatkin;given-names:NM surname:Smith;given-names:TJ surname:Gordon;given-names:JI 10.1074/jbc.R109.022848 19553672 REF J Biol Chem ref 284 2009 62569 Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm 86 90 surname:Bolam;given-names:DN surname:Sonnenburg;given-names:JL 10.4161/gmic.2.2.15232 21637023 REF Gut Microbes ref 2 2011 62660 Mechanistic insight into polysaccharide use within the intestinal microbiota e1000798 surname:Ellrott;given-names:K surname:Jaroszewski;given-names:L surname:Li;given-names:W surname:Wooley;given-names:JC surname:Godzik;given-names:A 10.1371/journal.pcbi.1000798 20532204 REF PLoS Comput Biol ref 6 2010 62737 Expansion of the protein repertoire in newly explored environments: human gut microbiome specific protein families 563 569 surname:Bolam;given-names:DN surname:Koropatkin;given-names:NM 10.1016/j.sbi.2012.06.006 22819666 REF Curr Opin Struct Biol ref 22 2012 62852 Glycan recognition by the Bacteroidetes Sus-like systems 625 641 surname:Zhou;given-names:Q surname:Rutland;given-names:MW surname:Teeri;given-names:TT surname:Brumer;given-names:H 10.1007/s10570-007-9109-0 REF Cellulose ref 14 2007 62909 Xyloglucan in cellulose modification 1532 1542 surname:Koropatkin;given-names:N surname:Martens;given-names:EC surname:Gordon;given-names:JI surname:Smith;given-names:TJ 10.1021/bi801942a 19191477 REF Biochemistry ref 48 2009 62946 Structure of a SusD homologue, BT1043, involved in mucin O-glycan utilization in a prominent human gut symbiont 3466 3475 surname:von Schantz;given-names:L surname:Håkansson;given-names:M surname:Logan;given-names:DT surname:Nordberg-Karlsson;given-names:E surname:Ohlin;given-names:M 10.1002/prot.24700 25302425 REF Proteins ref 82 2014 63058 Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Pi interactions 4799 4809 surname:Luís;given-names:AS surname:Venditto;given-names:I surname:Najmudin;given-names:S surname:Gilbert;given-names:HJ surname:Temple;given-names:MJ surname:Rogowski;given-names:A surname:Baslé;given-names:A surname:Xue;given-names:J surname:Knox;given-names:JP surname:Prates;given-names:JA surname:Ferreira;given-names:LM surname:Fontes;given-names:CM 10.1074/jbc.M112.432781 23229556 REF J Biol Chem ref 288 2013 63184 Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan 1205 1211 surname:Tsukimoto;given-names:K surname:Takada;given-names:R surname:Araki;given-names:Y surname:Suzuki;given-names:K surname:Karita;given-names:S surname:Wakagi;given-names:T surname:Shoun;given-names:H surname:Watanabe;given-names:T surname:Fushinobu;given-names:S 10.1016/j.febslet.2010.02.027 20159017 REF FEBS Lett ref 584 2010 63283 Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module 7224 7230 surname:Cho;given-names:KH surname:Salyers;given-names:AA 10.1128/JB.183.24.7224-7230.2001 11717282 REF J Bacteriol ref 183 2001 63363 Biochemical analysis of interactions between outer membrane proteins that contribute to starch utilization by Bacteroides thetaiotaomicron 40 49 surname:Rakoff-Nahoum;given-names:S surname:Coyne;given-names:MJ surname:Comstock;given-names:LE 10.1016/j.cub.2013.10.077 24332541 REF Curr Biol ref 24 2014 63502 An ecological network of polysaccharide utilization among human intestinal symbionts e00909-14 surname:Elhenawy;given-names:W surname:Debelyy;given-names:MO surname:Feldman;given-names:MF 10.1128/mBio.00909-14 24618254 REF mBio ref 5 2014 63587 Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles 94 108 surname:Hemsworth;given-names:GR surname:Déjean;given-names:G surname:Davies;given-names:GJ surname:Brumer;given-names:H 10.1042/BST20150180 26862194 REF Biochem Soc Trans ref 44 2016 63686 Learning from microbial strategies for polysaccharide degradation 43 60 surname:Noinaj;given-names:N surname:Guillier;given-names:M surname:Barnard;given-names:TJ surname:Buchanan;given-names:SK 10.1146/annurev.micro.112408.134247 20420522 REF Annu Rev Microbiol ref 64 2010 63752 TonB-dependent transporters: regulation, structure, and function e1002118 surname:Renzi;given-names:F surname:Manfredi;given-names:P surname:Mally;given-names:M surname:Moes;given-names:S surname:enö;given-names:P surname:Cornelis;given-names:GR 10.1371/journal.ppat.1002118 21738475 REF PLoS Pathog ref 7 2011 63817 The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG 499 511 surname:Phansopa;given-names:C surname:Roy;given-names:S surname:Rafferty;given-names:JB surname:Douglas;given-names:CW surname:Pandhal;given-names:J surname:Wright;given-names:PC surname:Kelly;given-names:DJ surname:Stafford;given-names:GP 10.1042/BJ20131415 24351045 REF Biochem J ref 458 2014 63929 Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species 1241 1252 surname:Sonnenburg;given-names:ED surname:Zheng;given-names:H surname:Joglekar;given-names:P surname:Higginbottom;given-names:SK surname:Firbank;given-names:SJ surname:Bolam;given-names:DN surname:Sonnenburg;given-names:JL 10.1016/j.cell.2010.05.005 20603004 REF Cell ref 141 2010 64088 Specificity of polysaccharide use in intestinal Bacteroides species determines diet-induced microbiota alterations e224 surname:Blanvillain;given-names:S surname:Meyer;given-names:D surname:Boulanger;given-names:A surname:Lautier;given-names:M surname:Guynet;given-names:C surname:Denancé;given-names:N surname:Vasse;given-names:J surname:Lauber;given-names:E surname:Arlat;given-names:M 10.1371/journal.pone.0000224 17311090 REF PLoS One ref 2 2007 64203 Plant carbohydrate scavenging through tonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria 669 677 surname:Gilbert;given-names:HJ surname:Knox;given-names:JP surname:Boraston;given-names:AB 10.1016/j.sbi.2013.05.005 23769966 REF Curr Opin Struct Biol ref 23 2013 64324 Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules 1251 1253 surname:Gordon;given-names:JI 10.1126/science.1224686 22674326 REF Science ref 336 2012 64448 Honor thy gut symbionts redux 1 7 surname:Hutkins;given-names:RW surname:Krumbeck;given-names:JA surname:Vaughan;given-names:E surname:Sanders;given-names:ME surname:Bindels;given-names:LB surname:Cani;given-names:PD surname:Fahey;given-names:G;suffix:Jr surname:Goh;given-names:YJ surname:Hamaker;given-names:B surname:Martens;given-names:EC surname:Mills;given-names:DA surname:Rastal;given-names:RA 10.1016/j.copbio.2015.09.001 26431716 REF Curr Opin Biotechnol ref 37 2016 64478 Prebiotics: why definitions matter 105 124 surname:Van Duyne;given-names:GD surname:Standaert;given-names:RF surname:Karplus;given-names:PA surname:Schreiber;given-names:SL surname:Clardy;given-names:J 10.1006/jmbi.1993.1012 7678431 REF J Mol Biol ref 229 1993 64513 Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin 43010 43017 surname:Freelove;given-names:AC surname:Bolam;given-names:DN surname:White;given-names:P surname:Hazlewood;given-names:GP surname:Gilbert;given-names:HJ 10.1074/jbc.M107143200 11560933 REF J Biol Chem ref 276 2001 64600 A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi surname:Holdeman;given-names:LV surname:Cato;given-names:ED surname:Moore;given-names:WEC REF Anaerobe laboratory manual ref 1977 64711 447 457 surname:Martens;given-names:EC surname:Chiang;given-names:HC surname:Gordon;given-names:JI 10.1016/j.chom.2008.09.007 18996345 REF Cell Host Microbe ref 4 2008 64712 Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:11:30Z glycan 307 326 surname:Otwinowski;given-names:Z surname:Minor;given-names:W REF Methods Enzymol ref 276 1997 64818 Processing of X-ray diffraction data collected in oscillation mode 582 601 surname:Terwilliger;given-names:TC surname:Adams;given-names:PD surname:Read;given-names:RJ surname:McCoy;given-names:AJ surname:Moriarty;given-names:NW surname:Grosse-Kunstleve;given-names:RW surname:Afonine;given-names:PV surname:Zwart;given-names:PH surname:Hung;given-names:LW 10.1107/S0907444909012098 19465773 REF Acta Crystallogr D Biol Crystallogr ref 65 2009 64885 Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard 61 69 surname:Terwilliger;given-names:TC surname:Grosse-Kunstleve;given-names:RW surname:Afonine;given-names:PV surname:Moriarty;given-names:NW surname:Zwart;given-names:PH surname:Hung;given-names:LW surname:Read;given-names:RJ surname:Adams;given-names:PD 10.1107/S090744490705024X 18094468 REF Acta Crystallogr D Biol Crystallogr ref 64 2008 64990 Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard 1948 1954 surname:Adams;given-names:PD surname:Grosse-Kunstleve;given-names:RW surname:Hung;given-names:LW surname:Ioerger;given-names:TR surname:McCoy;given-names:AJ surname:Moriarty;given-names:NW surname:Read;given-names:RJ surname:Sacchettini;given-names:JC surname:Sauter;given-names:NK surname:Terwilliger;given-names:TC 10.1107/S0907444902016657 12393927 REF Acta Crystallogr D Biol Crystallogr ref 58 2002 65095 PHENIX: building new software for automated crystallographic structure determination 2126 2132 surname:Emsley;given-names:P surname:Cowtan;given-names:K 10.1107/S0907444904019158 15572765 REF Acta Crystallogr D Biol Crystallogr ref 60 2004 65180 Coot: model-building tools for molecular graphics 658 674 surname:McCoy;given-names:AJ surname:Grosse-Kunstleve;given-names:RW surname:Adams;given-names:PD surname:Winn;given-names:MD surname:Storoni;given-names:LC surname:Read;given-names:RJ 10.1107/S0021889807021206 19461840 REF J Appl Crystallogr ref 40 2007 65230 Phaser crystallographic software 125 132 surname:Long;given-names:F surname:Vagin;given-names:AA surname:Young;given-names:P surname:Murshudov;given-names:GN 10.1107/S0907444907050172 18094476 REF Acta Crystallogr D Biol Crystallogr ref 64 2008 65263 BALBES: a molecular-replacement pipeline 56 66 surname:Tuomivaara;given-names:ST surname:Yaoi;given-names:K surname:O’Neill;given-names:MA surname:York;given-names:WS 10.1016/j.carres.2014.06.031 25497333 REF Carbohydr Res ref 402 2015 65304 Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature 24922 24933 surname:Martinez-Fleites;given-names:C surname:Guerreiro;given-names:CI surname:Baumann;given-names:MJ surname:Taylor;given-names:EJ surname:Prates;given-names:JA surname:Ferreira;given-names:LM surname:Fontes;given-names:CM surname:Brumer;given-names:H surname:Davies;given-names:GJ 10.1074/jbc.M603583200 16772298 REF J Biol Chem ref 281 2006 65449 Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation